Dear Jill,

thank you very much for your detailed answer to my question concerning the
detection of proteins in hydrogels. Since I am not yet that familiar with this
topic I still have some questions:

We want to know the enzyme distribution in alginate beads using a CLSM.
Unfortunately, the alginate is not distributed homogeneoulsy throughout the bead
radius due to the manufacturing process. Therefore, our guess is that the enzyme
is also not evenly distributed.

The alginate beads are made like the following. We have an alginate solution and
add the enzyme to this solution. Then the alginate is dropped into a Ca-solution
and the Ca ions cause the gel formation.

If I understood the given suggestions properly we have to basic methods of
detecting the proteins:

1) Before we add the enzyme to the alginate solution we can use a dye such as
Fluorescein oregon green. Then the dye is bound covalently to the enzyme.

2) Or: We could add a dye such as SYPRO Ruby protein gel stain to the alginate
solution. And here is what I am not sure about: As I mentioned before after the
bead formation the alginate is not evenly distributed anymore. So my guess is that
the dye will also be inhomogeneously distributed after the bead formation. So
then, even if we would have an evenly distributed enzyme concentration the
fluoresence intensity would be different.

What do you think about that? It would be greatly appreciated if you could give me
some hints if I am on the right track ...

Thanks in advance,

Matthias

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Dipl.-Ing. Matthias Heinemann    Aachen University of Technology (RWTH)
                                 Chair of Biochemical Engineering
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